Preventing biofilm assembly or facilitating biofilm dispersal would expedite treatment of biofilm-related infections. We hypothesize that penicillin-binding proteins (PBPs) are responsible for non-canonical D-amino acid incorporation into the peptidoglycan of Bacillus subtilis and, once incorporated, play a role in biofilm stability. Throug this proposed work, we expect to show that non-canonical D-amino acids, specifically D-Tyr, are incorporated into the pentapeptide of B. subtilis peptidoglycan in vivo using a combination of enzymatic degradation, chromatography, mass spectrometry, and comparison to chemically synthesized standards. We will also show that B. subtilis PBP 1a and 1b can perform this function in vitro by utilizing chemically synthesized lipid II substrate and cloned PBP1a and 1b from B. subtilis to assess their incorporation efficiency. Mutations in a single gene influence the disassembly of biofilms of Bacillus subtilis. We expect that recently identified mutations in ponA, the gene encoding for PBP 1a and 1b will give rise to mutant PBPs that are inefficient in incorporating D-Tyr into peptidoglycan. We will clone, over express, and purify the mutant B. subtilis PBPs and test incorporation efficiency to further validate our hypothesis that cell wall tailoring is responsible for biofilm regulation. This work will provide the foundation for elucidatng the mechanism of D- amino acid triggered biofilm disassociation. Demonstrating biofilm regulation by PBPs will allow for the development of new strategies toward treatment of biofilm related infections.